Synthetic approach to designed chemical structures

ABSTRACT

This invention relates to the chemical design and production of peptides, peptide structure and three dimensional conformation was assessed using NMR, circular dichroisin and pulsed field gradient NMR. In addition, this invention relates to peptides produced by these methods and to methods for using the peptides.

FIELD OF THE INVENTION

This invention relates to the fields of chemical design and to methodsfor selecting, modifying, and creating synthetic chemical structures andto their methods of use.

BACKGROUND

A critical feature of a polypeptide is its ability to fold into a threedimensional conformation or structure. Polypeptides usually have aunique conformation which, in turn, determines their function. Theconformation of a polypeptide has several levels of structure. Theprimary structure is a linear sequence of a series of amino acids linkedinto a polypeptide chain. The secondary structure describes the paththat the polypeptide backbone of the polypeptide follows in space, andthe tertiary structure describes the three dimensional organization ofall the atoms in the polypeptide chain, including the side groups aswell as the polypeptide backbone.

Covalent and noncovalent interactions between amino acids determine theconformation of a polypeptide. The most common covalent bond used inestablishing the secondary and tertiary structure of a polypeptide isthe formation of disulfide bridges between two cysteine residues(forming cysteine). The formation of noncovalent bonds is influenced bythe aqueous environment such as water. A large number of noncovalentinteractions, such as van der Waals, ionic, hydrophobic andhydrogen-bonded interactions, contribute to the way in which apolypeptide folds. Hydrophobic interactions, which occur between aminoacids with nonpolar side chains, are particularly important because theyassociate to exclude water. These side chains generally form the core ofthe polypeptide, where they are mostly inaccessible to water.

The secondary structure of polypeptides can be divided into two generalclasses: α-helix and β-sheet. An α-helix is stabilized by hydrogenbonding and side chain interactions between amino acids three and fourresidues apart in the same polypeptide chain, whereas a β-sheet isstabilized by hydrogen bonding and side chain interactions between aminoacids more distant in a polypeptide chain and in different polypeptidechains. A complete understanding of the construction of α helices and βsheets is important for the manipulation of the structure and functionof polypeptides.

A major challenge in de novo polypeptide design (more often referred toas de novo peptide design), which is the design of polypeptides (orpeptides) from scratch, is the engineering of a polypeptide having thefolding stability of the native structure of a natural polypeptide.Several polypeptides have been designed with the α helix as the majorstructural element. Few polypeptides have been designed with the β sheetas the major structural element. Unlike α helices where there is aregular succession of hydrogen bonds between amides three and fourresidues apart in the sequence, β sheets are formed by residues atvariable and often distant positions in the sequence. In addition, theytend to form aggregates in solution and precipitate under physiologicalconditions. A major difficulty in designing a structurally stable Bpolypeptide is dealing with the interactions between β sheets.

Designing a polypeptide to form a β-sheet has in the past usually beenbased on one of a number of structural propensity scales known in theart. These scales are derived either statistically from structuraldatabases of known folded polypeptides or by making single or minimalsite-specific changes in a fully folded polypeptide. See, for example,C. A. Kim, et al., Nature, 362, 267 (1993); D. L. Minor, et al., Nature,371, 264 (1994); D. L. Minor, et al., Nature, 367, 660 (1994); and C. K.Smith, et al., Biochemistry, 33, 5510 (1994). However, such scales aregenerally less useful when designing de novo β-sheet folds in shortpeptides where considerably more β-sheet and/or side-chain surface(particularly hydrophobic surface) will be exposed to water. D. E.Otzen, et al., Biochemistry, 34, 5718 (1995).

Betabellin was one of the first de novo designed class of β-sheetpeptides. J. Richardson, et al., Biophys. J., 63, 1186 (1992). It wasintended to fold into a sandwich of two identical four-stranded,antiparallel β sheets. A more recent version of betabellin, betabellin14D, was designed by Yan, et al., Protein Science, 3, 1069, (1994).Quinn, et al. designed betadoublet, which is similar to betabellin butcontains only naturally encoded amino acids. T. P. Quinn, et al., Proc.Natl. Acad. Sci. U.S.A., 91, 8747 (1994).

However, peptides in the betabellin and betadoublet series show limitedsolubility in water and minimal, highly transient β-sheet structure,i.e., nonstable structures. The best betabellin made thus far,Betabellin peptide 14D, for example, becomes less soluble at pH valuesabove 5.5 making it impractical for use at a physiological pH. Moreoverthe β-sheet structure formed by peptide 14D relies on the presence of anintermolecular disulfide bridge to yield a dimeric species. The peptidesof the present invention do not have these limitations. Betadoublet,which has the same predicted antiparallel β-sheet motif as betabellin,is even less water soluble, and only at a lower pH of about 4, and failsto show any compact, stable folding, i.e., structure.

Water solubility and pH ranges are important to peptide function. Apolypeptide that is not soluble under physiological conditions (i.e., inwater at a pH of about 7.0-7.4 and in about 150 mM NaCl or an equivalentphysiological salt) is not functional and is therefore not useful.Neither the betabellin nor the betadoublet strategies for peptide designachieved sufficient solubility, peptide compactness, or peptideself-association under physiological conditions.

Hence, there remains a need for β-sheet forming peptides that are notonly water soluble, but soluble at physiological conditions, and selfassociate.

Sepsis syndrome continues to be one of the leading causes of mortalityin critically ill patients and gram-negative bacterial pathogens causeabout ⅓ of these cases. Despite intensive laboratory and clinicalinvestigation, the mortality associated with gram-negative bacterialsepsis and shock remains at about 40%, a statistic that has changedlittle over time. Lipopolysaccharide (LPS, or endotoxin) is an integralcomponent of the outer membrane of gram-negative bacteria and triggersactivation of macrophages that, in turn synthesize and secrete cytokineswithin the endogenous tissue milieu and systemic circulation. Theresultant release of tumor necrosis factor-α (TNF-α) and other cytokinesby macrophages is causally linked to the host inflammatory response andthe subsequent development of septic shock. Unfortunately, standardinflammatory response and the subsequent development of sepsis andshock, including administration of potent antibiotics, aggressive fluidresuscitation, hemodynamic monitoring, and metabolic support, has notbeen associated with a significant reduction in mortality.

A 27 amino acid synthetic peptide based on amino acids 82-103 of BPIsignificantly inhibited TNF-α secretion in vitro and administration ofthe peptide in animal models diminished endotoxin levels, althoughabrogation of TNF-α secretion was incomplete (Battafarano et al. Surgery118:318-324, 1995 and Dahlberg, et al. J. Surg. Res. 63:44-48, 1996).The effect of anti-endotoxin monoclonal antibodies HA-1A and E5 onmortality during sepsis syndrome has been studied by phase III clinicaltrial. In these studies, mortality rates were not reduced as compared toplacebo treatments (The CHESS trial study group, Ann. Int. Med. 121:1-5,1994; Bone et al. Crit. Care Med. 23:994-1005, 1995). Accordingly, novelreagents are needed to treat gram-negative bacterial infections.

Tumor growth can be controlled by deprivation of vascularization (seeFolkman, Natl. Cancer. Inst. 82: 4-6, 1990 and Folkman et al. J. Biol.Chem. 267:10931-10934, 1992). A growing number of endogenous inhibitorsof angiogenesis include platelet factor-4 (PF4, Gupta et al. Proc. Natl.Acad. Sci. USA 92:7799-7803, 1995), interferon-γ inducible protein-10(IP-10, Luster, et al. J. Exp. Med. 182:219-231, 1995), as well assynthetic agents including thalidomide, metalloproteinase inhibitors,and the like. There is a need for reagents to inhibit angiogenesisincluding agents that inhibit endothelial cell proliferation for avariety of applications, including, but not limited to tumorigenesis.

SUMMARY OF THE INVENTION

The present invention provides a method for synthesizing a water-solublepeptide having at least about 35% amino acids having hydrophobic sidechains, the method comprising combining amino acids having charged sidechains and amino acids having noncharged polar side chains with aminoacids having hydrophobic side chains, wherein the amino acids havingcharged side chains are provided in a ratio of at least about 2:1 aminoacids having positively charged side chains to amino acids havingnegatively charged side chains.

The present invention also provides a method for synthesizing awater-soluble peptide having at least about 35% amino acids havinghydrophobic side chains, the method comprising combining amino acidshaving charged side chains and less than about 20% amino acids havingnoncharged polar side chains with amino acids having hydrophobic sidechains, wherein: the amino acids having charged side chains are providedin a ratio of at least about 2:1 amino acids having positively chargedside chains to amino acids having negatively charged side chains; thewater-soluble peptide has about 35% to about 55% amino acids havinghydrophobic side chains; and at least two of the amino acids havinghydrophobic side chains are positioned in the peptide with anintervening turn sequence in a manner such that the two amino acidshaving hydrophobic side chains are capable of aligning in a pairwisefashion to form a β-sheet structure; and the turn sequence is LXXGR,wherein each X is independently selected from the group consisting of K,N, S, and D. Herein, percentages are reported as the number of specifiedamino acids relative to the total number of amino acids in the peptidechain.

This invention also relates to a series of βpep peptides prepared usingthe methods of this invention. These peptides are provided as βpep-1through βpep-30 and correspond to SEQ ID NO: 1 through SEQ ID NO:30.This invention also relates to a method for treating a bacterialinfection or endotoxic shock comprising administering an amount of apharmaceutical composition effective to inhibit the bacterial infectionor neutralize endotoxin to a mammal, wherein the pharmaceuticalcomposition comprises (a) a peptide demonstrating bactericidal activityor endotoxin neutralizing activity selected from the group consisting ofβpep-1 through βpep30 (SEQ ID NO: 1 through SEQ ID NO:30); and (b) apharmaceutically acceptable carrier. In one embodiment, the peptideneutralizes endotoxin, in another the peptide is bactericidal and inanother the peptide is both bactericidal and neutralizes endotoxin. In apreferred embodiment, the peptide has endotoxin neutralizing activityand is selected from the group consisting of βpep-8 and βpep-23. Inanother preferred embodiment, the peptide has bactericidal activity andis selected from the group consisting of βpep-9, βpep-7, βpep-4, βpep-22and βpep-1.

This invention also relates to a method for inhibiting TNF-α levels in amammal comprising the step of administering a therapeutically effectiveamount of a pharmaceutical composition comprising: (a) a peptidedemonstrating bactericidal activity or endotoxin neutralizing activityselected from the group consisting of βpep-1 through βpep30 (SEQ ID NO:1 through SEQ ID NO:30); and (b) a pharmaceutically acceptable carrier.In a preferred embodiment the peptide is βpep-3.

This invention also relates to a method for inhibiting endothelial cellproliferation comprising the step of administering an effective amountof a composition comprising: a peptide demonstrating endothelial cellproliferation inhibition selected from the group consisting of βpep-1through βpep-30 (SEQ ID NO: 1 through SEQ ID NO:30). In one embodiment,the composition is a therapeutically effective amount of apharmaceutical composition comprising: a peptide selected from the groupconsisting of βpep-14 or βpep-16; and a pharmaceutically acceptablecarrier.

The invention also relates to a method for promoting inter-cellularadhesion molecule (ICAM) expression comprising the step of administeringan effective amount of a composition comprising: a peptide promotinginter-cellular adhesion molecule expression selected from the groupconsisting of βpep-1 through βpep-30 (SEQ ID NO:1 through SEQ ID NO:30).

“Amino acid” is used herein to refer to a chemical compound with thegeneral formula: NH₁₂—CRH—COOH, where R, the side chain, is H or anorganic group. Where R is organic, R can vary and is either polar ornonpolar (i.e., hydrophobic). The amino acids of this invention can benaturally occurring or synthetic (often referred to asnonproteinogenic). As used herein, an organic group is a hydrocarbongroup that is classified as an aliphatic group, a cyclic group orcombination of aliphatic and cyclic groups. The term “aliphatic group”means a saturated or unsaturated linear or branched hydrocarbon group.This term is used to encompass alkyl, alkenyl, and alkynyl groups, forexample. The term “cyclic group” means a closed ring hydrocarbon groupthat is classified as an alicyclic group, aromatic group, orheterocyclic group. The term “alicyclic group” means a cyclichydrocarbon group having properties resembling those of aliphaticgroups. The term “aromatic group” refers to mono- or polycyclic aromatichydrocarbon groups. As used herein, an organic group can be substitutedor unsubstituted. One letter and three letter symbols are used herein todesignate the naturally occurring amino acids. Such designationsincluding R or Arg, for Arginine, K or Lys, for Lysine, G or Gly, forGlycine, and X for an undetermined amino acid, and the like, are wellknown to those skilled in the art.

The term “peptide” or “polypeptide” is used herein to refer to an aminoacid polymer. A single peptide of this invention preferably has at least20 amino acids. Preferably the peptides of this invention are no greaterthan 50 amino acids in length, and more preferably about 28 to about 33amino acids in length.

The term “water-soluble” is used herein to refer to compounds,molecules, and the like, including the peptides of this invention, thatare preferably readily dissolved in water. The compounds of thisinvention are readily dissolved in water if about 1 mg of the compounddissolves in 1 ml of water having a temperature of about 35-45° C. Morepreferably, the peptides of this invention will have a water solubilityof at least about 10 mg/ml and often of at least about 20 mg/ml. Evenmore preferably, the peptides are soluble at these concentrations underphysiological conditions, including a pH of about 7.0-7.4 and a saltconcentration of about 150 mM NaCl.

The term “hydrophobic amino acid side chain” or “nonpolar amino acidside chain,” is used herein to refer to amino acid side chains havingproperties similar to oil or wax in that they repel water. In water,these amino acid side chains interact with one another to generate anonaqueous environment. Examples of amino acids with hydrophobic sidechains include, but are not limited to, valine, leucine, isoleucine,phenylalanine, and tyrosine.

The term “polar amino acid side chain” is used herein to refer to groupsthat attract water or are readily soluble in water or form hydrogenbonds in water. Examples of polar amino acid side chains includehydroxyl, amine, guanidinium, amide, and carboxylate groups. Polar aminoacid side chains can be charged or noncharged.

The term “noncharged polar amino acid side chain” or “neutral polaramino acid side chain” is used herein to refer to amino acid side chainsthat are not ionizable or do not carry an overall positive or negativecharge. Examples of amino acids with noncharged polar or neutral polarside chains includes serine, threonine, glutamine, and the like.

The term “positively charged amino acid side chain” refers to amino acidside chains that are able to carry a full or positive charge and theterm “negatively charged amino acid side chain” refers to amino acidside chains that are able to carry a negative charge. Examples of aminoacids with positively charged side chains include arginine, histadine,lysine, and the like. Examples of amino acids with negatively chargedside chains include aspartic acid and glutamic acid, and the like.

The term “self-association” refers to the spontaneous association of twoor more individual peptide chains or molecules irrespective of whetheror not the peptide chains are identical.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the alignment of β-sheet regions from the polypeptidesPF4, IL-8 and GRO polypeptides. β-sheet residues are blocked-in andlines connect the residues that are paired in the chain. The C-terminiin the sequences were synthesized in the amide form. Numbering shownbelow the PF4 sequence is that from native PF4.

FIG. 2 is a graph illustrating the far-ultraviolet circular dichroicspectra for designed peptides βpep-5, βpep-8, βpep-11 and βpep-1.Peptide concentration used was 10 to 20 μM in 20 mM potassium phosphate,pH=6.3. The temperature was varied from 5° C. to 75° C.

FIG. 3 is a graph depicting the ¹HNMR spectra for βpep-5, βpep-8,βpep-11 and βpep-1 in H₂O at 600 MHZ. Peptide concentration was 20 mg/mlin 20 mM potassium phosphate at a temperature of 40° C. and a pH of 6.3.Spectra were accumulated with 8,000 data points over 6000 Hz sweep widthand were processed with 3 Hz line broadening. Only the spectral regiondownfield from the HDO resonance is shown.

FIG. 4 is a graph showing pulsed-field gradient (PFG) NMR diffusioncoefficients as a function of temperature for βpep-5, βpep-8, βpep-11,βpep-1 and lysozyme.

FIG. 5 is a graph demonstrating the capacity of anti-endotoxin peptidesBG22 and βpep-3 to diminish LPS-induced secretion of TNF-α in vitro byRAW 264.7 cells.

FIG. 6 is a graph demonstrating the capacity of anti-endotoxin peptidesBG22 and βpep-3 to diminish TNF-α secretion during endotoxemia in vivo.Data are provided at 90 minutes post-challenge.

FIG. 7 provides ³H-Thymidine incorporation data for two different typesof endothelial cells with peptide (βpep-1 through βpep-24)concentrations of 2×10⁻⁶M. FIG. 7A provides ³H-Thymidine incorporationdata for FBHEC cells and FIG. 7B provides ³H-Thymidine incorporationdata for HUVEC cells.

FIG. 8A illustrates the P. aeruginosa bactericidal activity of βpep-1through βpep-24 at a peptide concentration of 1.2×10⁻⁷ M and FIG. 8Billustrates the endotoxin neutralizing activity of βpep-1 throughβpep-24 at a peptide concentration of 1.2×10⁻⁶ M.

DETAILED DESCRIPTION OF THE INVENTION

An intricate interplay exists between peptide β-sheet formation,self-association, and water solubility. A challenge in making a solublefolded peptide is that solubility has a double-edged effect:precipitation versus over-solvation. Precipitation is the falling out ofsolution of a peptide, while over-solvation is the tendency of a solublepeptide to prefer intermolecular water-peptide interactions overintramolecular folding interactions. Going too far in either direction(precipitation or over-solvation) destabilizes the folded state. Reducedsolubility generally occurs due to intermolecular peptide-to-peptideinteractions (hydrophobic and electrostatic) which results inprecipitation or gelation. Although the precipitate, for example, is inequilibrium with soluble peptide, the equilibrium is shifted away fromsolution. If a designed β-sheet-forming peptide contains a relativelylarge number of amino acids with hydrophobic side chains which are notscreened to some extent by the folding process, precipitation orgelation may result. Inherent in the design of β-sheet forming peptides,therefore, is the capacity to self-associate, thereby screeninghydrophobic surface from solvent water.

The present invention provides a method for the de novo design ofpeptides that are water soluble at or near physiological conditions andpreferably form β-sheet structures. Preferably and advantageously, thewater-soluble peptide forms, through self-association, a β-sheet in theabsence of any intermolecular covalent interactions (although this isnot necessarily a requirement). The method takes into account thefollowing parameters: the number or percentage composition of aminoacids with positively and negatively charged side chains, the number orpercentage composition of amino acids with noncharged polar side chains,the number or percentage composition of amino acids with hydrophobicside chains, proper placement and pairing of amino acids in the sequenceand in space, and specific turn character. The specific turn characterrefers to the composition of side chains of the amino acids positionedin the turn sequence. A turn sequence refers to a sequence of aminoacids that reverses the direction of the amino acid sequence in space.

When the number of amino acids with positively and negatively chargedside chains is about equal, intermolecular electrostatic interactionsshift the solvation-precipitation equilibrium to the precipitate state.This was a fundamental flaw in previous design approaches. Adjusting theoverall net charge of the peptide to mostly amino acids with positivelycharged side chains greatly improves solubility. Inter-peptide chargerepulsion may also help to reduce precipitation. In a preferredembodiment of this invention, the ratio of amino acids with positivelycharged side chains to amino acids with negatively charged side chainsis at least about 2:1. Preferably the ratio of amino acids withpositively charged side chains to amino acids with negatively chargedside chains is no greater than about 3:1; however, this invention alsoconsiders larger ratios of amino acids with positively charged sidechains to amino acids with negatively charged side chains including, butnot limited to 4:1, 5:1, 6:1 or greater.

The other side to solubility is that a peptide can be too soluble, i.e.,over-solvated. When the number of amino acids with polar side chains istoo high and other stabilizing forces are too low, intramolecularcollapse or folding may be opposed by intermolecular peptide-waterassociations. Therefore, a high content of amino acids with short chainpolar side chains such as serine and threonine (the hydroxylated aminoacids) is not desirable, even though threonine is at the top of β-sheetpropensity scales. This was another fundamental flaw in previous designapproaches. The peptides of the present invention preferably containless than 100%, preferably less than about 50%, more preferably, lessthan about 20% amino acids with noncharged polar side chains.

An appropriate percent composition of amino acids with hydrophobic sidechains and proper placement in the sequence of such amino acids promotesself-association-induced structural collapse and stability. Thetrade-off is to adjust the percent composition of amino acids withhydrophobic side chains to avoid insolubility, while promoting foldingand structure formation. The peptides of this invention preferablycontain about 35% to about 55% amino acids with hydrophobic side chains,and in particularly preferred embodiments, about 40% to about 50% aminoacids with hydrophobic side chains. In preferred embodiments of thisinvention, the hydrophobic amino acids, or combination thereof, arealiphatic, although aromatic hydrophobic amino acids are also possible.

To generate a more compact fold, side-chain pairing and packing must beoptimized. Hydrophobic interactions increase folded state stability.Choosing the proper placement of amino acids with hydrophobic sidechains in the sequence and combination of hydrophobic side-chaintriplets across the strands as well as between strands in theself-associated peptide is an important feature to designing stableβ-sheet folds. As used herein, a strand is that portion of a foldedpeptide chain between turn sequences.

Preferably, the amino acids are spacially positioned in the foldedpeptide to form a substantially hydrophobic surface. More preferably,the amino acids are spacially positioned in the folded peptide such thatone peptide molecule is capable of self-associating with another peptidemolecule to form a multimer.

The added dimension to this β-sheet design process is oligomerizationwhere efficient hydrophobic side-chain packing of one sheet on top ofanother appears to be important for optimum folding stability andcompactness. Choosing the proper placement of side chains, particularlyhydrophobic side chains, in the amino acid sequence is important tocontrolling fold stability. Compact β-sheet folding is typicallydependent on well-packed inter-strand side chain pairings. In apreferred embodiment of this invention at least two amino acids withhydrophobic side chains, and more preferably, three amino acids withhydrophobic side chains are positioned to align in space to form aβ-sheet structure. Between these amino acids are turn sequences to allowfor these side chain pairings.

Specific turn character may promote or stabilize a desired fold. Avariety of turn sequences are known in the art. For a particular β-sheetfold, some turns may be important, while others may not. Those skilledin the art will be able to incorporate a turn sequence into the peptidesdesigned according to the methods of this invention to test whether ornot the peptide maintains a β-sheet structure, and the like, followingthe methods provided in the Examples that follow. A specific novelfolding initiation turn/loop sequence, KXXGR (Ilyina et. al.,Biochemistry 33, 13436 (1994) was used in SEQ ID NOS:1-4 (βpep-5,βpep-8, βpep-11 and βpep-1), as described in the Example section of thisdisclosure. In this sequence, each X is independently selected from thegroup consisting of K, N, S, and D. This sequence was positioned betweentwo amino acids with hydrophobic side chains such that the two aminoacids having hydrophobic side chains were capable of aligning in apairwise fashion to form a β-sheet structure.

Using the invention disclosed herein, 30 peptides, βpep-1 throughβpep-30, were designed de novo (see Table 1, below). All βpep peptidesare water soluble at least up to 30 mg/mL (9 mM) at pH values betweenpH=2 and pH=10, and all form β-sheets and have been shown by circulardichoism (CD) and nuclear magnetic resonance (NMR) to form significantpopulations of self-association-induced β-sheet structure in water atnear-physiological conditions. βpep-1, in particular, exemplifies anexceptional application of this design approach by showing relativelystable, compact triple-stranded β-sheet structure with good side-chainpacking. Others which behave similarly are βpep4 through βpep-30.

Using the methods of this invention it is possible to create any numberof peptides, preferably peptides having β-sheet structure. Thisinvention provides a method for designing a peptide scaffold to supporta peptide in its native β-sheet structure. Those skilled in the art willrecognize that there are a variety of proteins and polypeptides withβ-sheet structures and that many of the proteins and polypeptidescontaining these β-sheet structures are known to mediate, promote orinhibit a variety of biological effects.

For example, a number of biological effects have been mapped to peptidesequences in a protein that exhibit β-sheet conformations. Thisinvention permits the selection of a peptide sequence from a protein ina domain having β-sheet structure and the incorporation of this peptidesequence into a scaffold, according to this invention, to create apeptide with retained β-sheet structure. These novel peptides can havethe same or improved biological activity that is attributed to thepeptide domain while in the native protein with the advantage that theremaining portions of the protein are not required for activity. The useof an entire protein to duplicate the effect of a peptide domaincomplicates experimentation and therapy. Other domains in a protein maystimulate other biological and chemical effects including, but notlimited to, enhanced antigenicity, decreased solubility, or stimulateunwanted biological responses, and the like. The protein is often labileor is not suited for biological or therapeutic applications withoutfurther modification. The strategy of this invention permits afunctional peptide domain to be retained in its tertiary conformationwithin the scaffold of this invention. The methods of this inventionprovide direction for those skilled in the art to generate a variety ofpeptides with novel activities or with activities attributable to aparent protein from which the peptide was originally derived.

Peptides once prepared using the methods of this invention can befurther modified in a variety of ways to form derivatives or analogs.These modifications include addition, substitution or deletion of aminoacids either before the peptides are tested for biological activity orafter testing the peptides for activity. Amino acid substitutionspreferably do not eliminate the biological activity of the peptide.Conservative amino acid substations typically can be made withoutaffecting biological activity. Conservative amino acid substitutionsinclude substitution of an amino acid for another amino acid of the sametype. Types of amino acids include: (1) basic amino acids such aslysine, arginine, and histidine; (2) hydrophobic amino acids such asleucine, isoleucine, valine, phenylalanine, and tryptophan; (3)non-polar amino acids including alanine, valine, leucine, isoleucine,proline, and methionine; (4) polar amino acids such is serine,threonine, cysteine, tyrosine, asparagine and glutamine; and (5)positively charged amino acids such as aspartic and glutamic acid.

The peptides prepared and designed according to this invention can beadministered alone in a pharmaceutically acceptable buffer, as anantigen in association with another protein, such as animmunostimulatory protein or with a protein carrier such as, but notlimited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),ovalbumin, or the like. Peptides can be conjugated to other proteinusing standard methods such as activation of the carrier molecule with aheterobifunctional sulfosuccinimidyl4-(n-maleimidomethyl)cyclohexane-1-carboxylate reagent. Cross-linking ofactivated carrier to a peptide can occur by reaction of the maleimidegroup of the carrier with the sulfhydryl group of a peptide containing acysteine residue. Conjugates can be separated from free peptide throughthe use of gel filtration column chromatography or other methods knownin the art.

The peptides with demonstrated biological activity can be administeredto a mammal in an amount alone or together with other active agents andwith a pharmaceutically acceptable buffer. For example, the peptidesprepared according to this invention that demonstrate endotoxinneutralizing activity and/or bactericidal activity can be administeredto treat a bacterial infection. The pharmaceutical composition caninclude a bactericidal peptide and/or an endotoxin neutralizing peptideprepared according to this invention. The peptides can be combined witha variety of physiological acceptable carriers for delivery to a patientincluding a variety of diluents or excipients known to those of ordinaryskill in the art. For example, for parenteral administration, isotonicsaline is preferred. For topical administration a cream, including acarrier such as dimethylsulfoxide (DMSO), or other agents typicallyfound in topical creams that do not block or inhibit activity of thepeptide can be used. Other suitable carriers include, but are notlimited to alcohol, phosphate buffered saline and other balances saltsolutions.

The peptides prepared according to this invention that demonstratebiological activity can be administered in a variety of ways, includingintravenously topically, orally and intramuscularly to a variety ofmammals, including humans, mice and rabbits. The peptides can beadministered as a single dose or in multiple doses. Preferably the doseis an effective amount as determine by the standard methods describedherein and includes about 1 microgram to about 1,000 microgramspretreatment, more preferably about 50 to about 250 microgramspretreatment and those skilled in the art of clinical trials will beable to optimize dosages of particular peptides through standard trialstudies.

The effective amount of a peptide for treating a bacterial infectionwill depend on the bacterial infection, the location of the infectionand the peptide. An effective amount of the peptide is that amount thatdiminishes the number of bacteria in the animal and that diminishes thesymptoms associated with bacterial infection such as fever, pain andother associated symptoms of the bacterial infection. The effectiveamount of a peptide can be determined by standard dose response methodsin vitro and an amount of peptide that is effective to kill at leastabout 50 to 100% of the bacteria (LD₅₀) and more preferably about 60 to100% of the bacteria would be considered an effective amount.

Alternatively, an effective amount of the peptide for treating abacterial infection can be determined in an animal system such as amouse. Acute peritonitis can be induced in mice such as outbred Swisswebster mice by intraperitoneal injection with bacteria such as P.aeruginosa as described by Dunn et al. (Surgery, 98:283, 1985); Cody etal. (Int. Surg. Res., 52:315, 1992). Different amounts of peptide can beinjected at one hour intravenously prior to the injection of thebacteria. The percentage of viable bacteria in blood, spleen and livercan be determined in the presence and absence of the peptide or otherantibiotics. While not meant to limit the invention, it is believed thatbactericidal peptide could also enhance the effectiveness of otherantibiotics such as erythromycin, and the like.

Peptides with endotoxin neutralizing activity can be used to treatmammals infected with gram-negative bacteria systemically and thatexhibit symptoms of endotoxin shock such as fever, shock and TNF-αrelease. The animals are typically infected with one or moregram-negative bacteria such as Pseudomonas spp., rough strains of E.coli, encapsulated E. coli and smooth strain E. coli. The endotoxinneutralizing peptide can be combined with other agents that are knownand used to treat endotoxin shock.

A number of exemplary peptides were designed according to the methods ofthis invention (βpep-1 through βpep-30) and the peptides weresynthesized according to Example 1. A number of these peptides appear tohave a variety of biological applications. In one example, a number ofpeptides prepared according to this invention had endotoxin neutralizingactivity, bactericidal activity or both endotoxin neutralizing activityand bactericidal activity.

A number of the peptides demonstrated endotoxin neutralizing activity,as compared to PF4 (see FIG. 1), and had improved activity as comparedto an equivalent region from protein B/PI (amino acids 86-108). Resultsof these studies are provided in Example 5, Example 6 and includingFIGS. 5 and 6. Domains within amino acids 86-108 of B/PI have been shownto have endotoxin neutralizing activity and the results from the studiesindicated that a number of β peptides had endotoxin neutralizingactivity. Endotoxin neutralizing activity can be measure by determiningthe molar concentration at which the peptide completely inhibits theaction of lipopolysaccharide in an assay such as the Limulus amoebocytelysate assay (LAL, Sigma Chemicals, St. Louis, Mo.) or the chromogenicLAL 1000 test (Biowhittacker, Walkersville, Md.). Endotoxin neutralizingactivity can also be measured by calculating an inhibitory dose 50(LD₅₀) using standard dose response methods. An inhibitory dose 50 isthat amount of peptide that can inhibit 50% of the activity ofendotoxin. Endotoxin activity can also be measured by determining theamount of release of tumor necrosis factor alpha (TNF-α) from amacrophage cell line or by evaluating the symptoms of shock in animals.Production of TNF-α can be assayed as described by Mossman et al.(Immunological Methods 65:55, 1983). Peptides preferably neutralizedendotoxin at a molar concentration of about 1×10⁻⁴ M to about 10⁻⁸M,more preferably about 10⁻⁵M to about 10⁻⁶M. Peptides were considered tonot have endotoxin neutralizing activity did not neutralize endotoxin ata molar concentration of 10⁻⁴ or less.

Peptides having biological activity have a size about 10 amino acids toabout 100 amino acids, more preferably about 10 to about 50 amino acids.Peptides with about 20 to about 50 amino acids are preferred andpeptides of 28-33 amino acids are particularly preferred.

A number of the peptides prepared by this invention had bactericidalactivity. Bactericidal activity can be evaluated against a variety ofbacteria, preferably gram negative bacteria, but the types of bacteriacan include Pseudomonas spp including P. aeruginosa and P. cepacia, E.coli strains, including E. coli B, Salmonella, Proteus mirabilis andStaphylococcus strains such as Staphylococcus aureus. A preferredorganism is Pseudomonas aeruginosa. Bactericidal activity is determinedby identifying the effective dose for killing as the molar concentrationof the peptide which results in at least a 60% killing of the bacteria,as determined by standard methods. Preferably, the peptide has aneffective dose at a concentration of about 1×10⁻⁴ M to about 1×10⁻¹⁰M,and more preferably 1×10⁻⁷M to about 1×10⁻⁹M. Peptides that were notconsidered to be bactericidal did not kill P. aeruginosa atconcentrations of 10⁻⁴M or less at a pH of 5.6. Bactericidal activitycan also be determined by calculating a lethal dose 50 (LD₅₀) usingstandard methods. The LD₅₀ is that amount of peptide or protein thatkills 50% of the bacteria when measured using standard dose responsemethods. A bactericidal peptide preferably has an LD_, of about 10⁻⁴ Mto about 10⁻⁹ M, more preferably about 10⁻⁷ M to about 10⁻⁹M.

In view of these results, this invention also relates to the use ofpeptides generated according to the methods of this invention todemonstrate bactericidal and/or endotoxin neutralizing activity. Atleast one of the peptides was also able to neutralize the effects ofTNF-α in vivo.

In addition, some of the peptides prepared according to the methods ofthis invention are capable of inhibiting angiogenesis and/or endothelialcell proliferation. Example 8 provides methods for testing theangiogenesis or endothelial proliferation inhibiting capacity ofpeptides prepared according to the methods of this invention. In apreferred embodiment, βpep4 was found to fold compactly and its NMRsolution structure comprised two differently aligned, six strandedanti-parallel β-sheet dimer amphipaths sandwiched to form a tetramer.The βpep-4 β-sheet sandwich tetramer has a highly positively chargedsurface which makes it and other βpep peptides (Mayo et al. Protein Sci5:1301-1315, 1996) good candidates for binding to anionic biomoleculeslike heparin and cell surface heparin sulfate and possibly formodulating various cellular activities. PF4 and other α-chemokines onwhich the βpep design was based, also have a relatively high netpositive surface charge, bind to polysulfated glycosaminoglycans likeheparin and trigger various cellular activities. Indeed, βpep-4 andrelated homologs may be novel chemokines. For this reason, a library ofβpep peptide 33 mers, along with parent PF4, was screened foranti-angiogenic and related activities as provided in Example 8.

The methods of this invention relate to the use of effective amounts ofthe peptides prepared according to this invention to treat bacterialinfection, endotoxic shock, mediate the effect of TNF-α (Example 7),inhibit endothelial cell proliferation and upregulate ICAM expression(Example 8). The compositions comprising the peptides of this inventioncan be added to cells in culture or used to treat mammals. Where thepeptides are used to treat mammals, the peptide is combined in apharmaceutical composition comprising a pharmaceutically acceptablecarrier such as a larger molecule to promote peptide stability or apharmaceutically acceptable buffer that serves as a carrier for thepeptide.

The invention will be further described by reference to the followingdetailed examples. These examples are offered to further illustrate thevarious specific and preferred embodiments and techniques. It should beunderstood, however, that many variations and modifications may be madewhile remaining within the scope of the present invention.

EXAMPLE 1 The Design and Synthesis of Water-Soluble β-Sheet FormingPeptides

Peptides of 33 amino acid residues in length, were synthesized on aMilligen Biosearch 9600 automated peptide synthesizer. The proceduresused were based on Merrifield solid phase synthesis utilizing Fmoc-BOPchemistry (Stewart et al., 1984. Solid phase peptide synthesis. 2nd ed.Rockford, Ill., Pierce Chemical Co. pp. 125-135). After the sequence hadbeen obtained, the peptide support and side chain protection groups wereacid cleaved (trifluoroacetic acid and scavenger mixture). Crudepeptides were analyzed for purity on a Hewlett-Packard 1090M analyticalHPLC using a reverse phase C18 VyDac column. Peptides generally wereabout 90% pure. Further purification was done on a preparativereverse-phase HPLC C-18 column using an elution gradient of 0-60%acetonitrile with 0.1% trifluoroacetic acid in water. Peptides then wereanalyzed for amino acid composition on a Beckman 6300 amino acidanalyzer by total hydrolysis of samples using 6N HCl at 110° C. for18-20 hrs. N-terminal sequencing confirmed peptide purity. The aminoacid sequences and compositions of the four peptides are given in Table1, and the composition of the peptides is provided in Table 2. βpep-11contains an amino acid in the D configuration as represented by thesuperscripted D.

table 1 Amino Acid Sequcnce of β-Peptides β pep-5 (SEQ ID NO:1)KFIVTLRVIKAGPHSPTAQIIVELKNGRKLSLD β pep-8 (SEQ ID NO:2)ANIKLSVEMKLFKRHLKWKIIYKLNDGRELSLD β pep-11 (SEQ ID NO:3)ANIKLSVEMKLFCY^(D)WKVCKIIVKLNDGRELSLD β pep-1 (SEQ ID NO:4)SIQDLNVSMKLFRKQAKWKIIVKLNDGRELSLD β pep-2 (SEQ ID NO:5)ANIKLSVKWKAQKRFLKMSINVDLSDGRELSLD β pep-3 (SEQ ID NO:6)HIKELQVKWKAQKRFLKMSIIVKLNDGRELSLD β pep-4 (SEQ ID NO:7)SIQDLNVSMKLFRKQAKWKINVKLNDGRELSLD β pep-6 (SEQ ID NO:8)HIKELQVRWRAQKRFLRMSIIVKLNDGRELSLD β pep-7 (SEQ ID NO:9)HIKELQVKMKAQKRFLKWSIIVKLNDGRELSLD β pep-9 (SEQ ID NO:10)ANIKLSVKWKAQKRFLKMSIIVKLNDGRELSLD β pep-10 (SEQ ID NO:11)ANIKLSVEMKLFCRHLKCKIIVKLNDGRELSLD β pep-12 (SEQ ID NO:12)ANIKLSVEMKFFKRHLKWKIIVKLNDGRELSLD β pep-13 (SEQ ID NO:13)ANIKLSVEFKLFKRHLKWKIIFKLNDGREFSLD β pep-14 (SEQ ID NO:14)SIQDLNVSMKLFRKQAKWKLIVKLNDGRELSLD β pep-15 (SEQ ID NO:15)SIQDLNVSMKLFRKQAKWKIILKLNDGRELSLD β pep-16 (SEQ ID NO:16)SIQDLNVSMKLFRKQAKWKIIAKLNDGRELSLD β pep-17 (SEQ ID NO:17)SIQDLNVSMKLFRKQAKWKILVKLNDGRELSLD β pep-18 (SEQ ID NO:18)SIQDLKVSMKLFRKQAKWKIIVKLNDGRELSLD β pep-19 (SEQ ID NO:19)SIQKLNVSMKLFRKQAKWKIIVKLNDGRELSLD β pep-20 (SEQ ID NO:20)SIQDLNVSMXLFRKQAKWKIIVKLNDGRELSLD“X” in this sequence refers to the noncommon aminoacid norleucine

β pep-21 (SEQ ID NO:21) SIQDLNVSLKLFRKQAKWKIIVKLNDGRELSLD β pep-22 (SEQID NO:22) SIQDLNLSMKLFRKQAKWKIIVKLNDGRELSLD β pep-23 (SEQ ID NO:23)SIQDLKVSLNLFRKQAKWKIIVKLNDGRELSLD β pep-24 (SEQ ID NO:24)SIQFLKVSLNLDRKQAKWKIIVKLNDGRELSLD β pep-25 (SEQ ID NO:25)ANIKLSVQMKLFKRHLKWKIIVKLNDGRELSLD β pep-26 (SEQ ID NO:26)SIQDLNVSMKLFRKQAKWKIIIKLNDGRELSLD β pep-27 (SEQ ID NO:27)SIQDLNVSMKLFRKQAKWKAIVKLNDGRELSLD β pep-28 (SEQ ID NO:28)SIQDLNVSMKLFRKQAKWKVIVKLNDGRELSLD β pep-29 (SEQ ID NO:29)SIQDLNVSMKLFRKQAKWKLILKLNDGRELSLD β pep-30 (SEQ ID NO:30)SIQDLNVSMKLFRKQAKWKVIIKLNDGRELSLD

Synthesized peptides used portions of human PF4 residues 22 to 54, IL-8residues 22 to 54, and Gro-α residues 24 to 56. The C-terminal asparticacid residue Cα carboxylate was made in the amide form. To avoidpotential problems with cysteine oxidation, cysteines found in nativesequences were replaced by serines. The native sequences for the PF4(Deuel et al., Proc. Natl. Acad. Sci. USA 74:2256-2258, 1977), IL-8(Holt, et al. Sem. Hematol. 22:151-163, 1985) and GR0 (Anisowicz et al.,Proc. Natl. Acad. Sci. USA 84:7199-7192, 1987) peptide fragments areprovided in FIG. 1

βpep-5 was composed primarily of sequences from the α-chemokinesplatelet factor 4 (PF4) and interleukin-8 (IL-8). Turn/loop 1 and 2 arefrom PF4. Turn 2 is the PF4 peptide folding initiation site. The threestrands were mostly from IL-8 with a few variations. The N-terminaldipeptide VT was included to create a potential strand 1-2 hydrophobicV25-L45 pair; a threonine was placed at position 26 to increase β-sheetpropensity and to maintain solubility. IL-8 peptide K44 and E50positions were switched to be more like PF4 with K50 and to maintain anyfolding stability from a potential salt-bridge pair.

βpep-8, βpep-11 and βpep-1 used a combination of α-chemokine sequences(primarily from strands 2 and 3 with the loop/turn initiation sequenceand alternative sequences in the first turn/loop and strand 1 which werederived in part from another polypeptide from this lab. Similar β-sheetalignments found in α-chemokine polypeptides PF4, IL-8 and growthrelated polypeptide (Gro-α) also were generally conserved in all fourpeptides. βpep-8 has the IL-8 sequence I41-D54 (IIVKLSDGRELSLD) with theremainder of the sequence derived from the β-sheet domain ofbactericidal/permeability increasing polypeptide. Three N-terminalresidues 24, 26 and 28 from strand 1 were replaced with I, L, V forproper hydrophobic pairings with strand 2 residues. The number ofresidues in turn 1 was increased by one, thereby shifting the N-terminalnumbering for paired residues by one relative to the α-chemokines.

βpep-11 differed from βpep-8 only in the first turn/loop, residues34-39. In βpep-11, a tight turn motif containing a D-tryptophan, i.e.,Y^(D)WKV, from somatostatin was used. Although two cysteines (for futureoxidation to cysteine) were also present in βpep-11 as in somatostatin,they were maintained in the reduced state (10⁻⁴ M perdeuteratedmercaptoethanol was added to the solution). βpep-1 was based on βpep-8with residues L26, V28, M30-F33 and K38-D54 being the same. The βpep-1N-terminus (S22-S29) was taken from another α-chemokine polypeptide,neutrophil activating peptide-2 (NAP-2), and turn/loop residues 34-37were conservative substitutions from those in βpep-8.

TABLE 2 Amino acid compositions of β-sheet-forming peptides. βpep5 βpep8βpep11 βpep1 (3582)¹ (3969)¹ (3839)¹ (3859)¹ HYDROPHOBIC Ile 4 3 3 3 Leu4 6 5 5 Val 3 2 3 2 Ala 2 1 1 1 Met — 1 1 1 Phe 1 1 1 1 Tyr — — 1 — Trp— 1 1 1 Pro 2 — — — TOTAL 16  15  16  14  HYDROPHOBIC Gly 2 1 1 1CHARGED Asp(−) 1 2 2 3 Glu(−) 1 2 2 1 Lys(+) 4 6 5 5 Arg(+) 2 2 1 2His(+) 1 1 — — NONCHARGED POLAR Asn 1 2 2 2 Gln 1 — — 2 Thr 2 — — — Ser2 2 2 3 Cys — — 2 — TOTAL POLAR 15  17  16  18  (charged plusnoncharged) ¹calculated molecular weight of peptide.

EXAMPLE 2 Circular Dichroism (CD) of the β-Peptide Series

Circular dichroism (CD) is one way to measure formation of a β-sheetstructure. CD spectra were measured on a JASCO JA-710 (Jasco, Eastern,Md.) automatic recording spectropolarimeter coupled with a dataprocessor. Curves were recorded digitally and fed through the dataprocessor for signal averaging and baseline subtraction. Spectra wererecorded from 5° C. to 65° C. in the presence of 10 mM potassiumphosphate, over a 185 nm to 250 nm range using a 0.5 mm path-length,thermally-jacketed quartz cuvette. Temperature was controlled by using aNesLab water bath. Peptide concentration was varied from 0.014 to 0.14mM. The scan speed was 20 nm/min. Spectra were signal-averaged 8 times,and an equally signal-averaged solvent baseline was subtracted. Theseexperiments are well known in the art.

For βpep-5, CD data resembled those observed for β-sheet-forming PF4peptide. Based on CD data alone, βpep-5 appeared not to form anyβ-sheets. CD data for all β peptides except βpep-5 showed a prominentband at 217 nm indicating formation of β-sheet structure. These peptideswere composed mostly of β-sheet structure. For βpep-1, it was surprisingthat the normally “random coil” 204-208 nm CD band was also prominentgiven NMR results, which indicated that βpep-1 formed one of the moststable and compact β-sheet structure of all 30 peptides. It may be thatthis band, which shifts from 204 nm to 208 nm as the 217 nm β-sheet bandbecomes more negative, was the result of stable turn structure.

To demonstrate that temperature modulated β-sheet folding in these βpeppeptides, the molar ellipticity at 217 nm for βpep-8, βpep-11 and βpep-1was plotted versus temperature as seen in FIG. 2. As the temperatureincreased from 5° C., the 217 nm band became more negative (especiallyfor βpep-11 and βpep-1) and leveled off between 35° C. and 50° C.,indicating an increase in the β-sheet population. This “cold melt”demonstrated a role for the hydrophobic effect in stabilizing β-sheetconformational populations. For βpep-8, in particular, the 217 nm bandbecame much more positive as the temperature increased further. Thiseffect reflected a more traditional structural “melt.” For βpep-11 andβpep-1, however, temperature increases up to 65° C. showed less of aneffect on the β-sheet population. These data suggested that β-sheets inβpep-11 and βpep-1 are relatively more “stable” than in βpep-8. βpep-14through βpep-30 also formed relatively stable compact β-sheets.

Those of ordinary skill in the art will readily recognize that theanalyses performed for βpep-5, βpep-8, βpep-11 and βpep-1 can readily beperformed for all 30 of the βpep peptides of this invention as well asany number of peptides prepared using the methods of this invention.

EXAMPLE 3 Nuclear Magnetic Resonance (NMR) of the β-Peptide Series

Since CD data indicate maximal β-sheet formation at about 40° C., NMRspectra (FIG. 3) were accumulated for βpep-5, βpep-8, βpep-11 and βpep-1and the other peptides at pH 6.3, 20 mM NaCl and 40° C. For NMRmeasurements, freeze-dried peptide was dissolved either in D₂O or inH₂O/D₂O (9:1). Polypeptide concentration normally was in the range of 1to 5 mM. pH was adjusted by adding UL quantities of NaOD or DCl to thepeptide sample. NMR spectra were acquired on a Bruker AMX-600 (BrukerInstrument, Inc., Bruker, Mass.) or AMX-500 NMR spectrometer. Forresonance assignments, double quantum filtered COSY (Piantini et al. J.Am. Chem. Soc. 104:6800-6801, 1982) and 2D-homonuclear magnetizationtransfer (HOHAHA) spectra, obtained by spin-locking with a MLEV-17sequence with a mixing time of 60 ms, were used to identify spin systems(See Bax, et al. J. Magn. Reson. 65: 355-360, 1985). NOESY experiments(see Jeener et al., J. Chem. Phys. 71:4546-4553, 1979 and Wider et al.J. Magn. Reson. 56:207-234, 1984) were performed to sequentially connectspin systems and to identify NOE connectivities. All 2D-NMR spectra wereacquired in the TPPI (Marion & Wuthrich, Biochem. Biophys, Res. Comun.113:967-974, 1983) or States-TPPI (States et al. J. Magn. Reson.48:286-292, 1982) phase sensitive mode. The water resonance wassuppressed by direct irradiation (0.8 s) at the water frequency duringthe relaxation delay between scans as well as during the mixing time inNOESY experiments. These experiments are well-known in the art.

2D-NMR spectra were collected as 256 to 400 tl experiments, each with 1k or 2 k complex data points over a spectral width of 6 kHz in bothdimensions with the carrier placed on the water resonance. For HOHAHA(COSY) and NOESY spectra, normally 16 and 64 scans, respectively, weretime averaged per tl experiment. The data were processed directly on theBruker AMX-600X-32 or offline on a Bruker Aspect-1 workstation with theBruker UXNMR program. Data sets were multiplied in both dimensions by a30-60 degree shifted sine-bell function and zero-filled to 1 k in the tldimension prior to Fourier transformation.

NMR data indicated that βpep-1 was best of βpep-5, βpep-8, βpep-11 andβpep-1 at forming a compact, triple-stranded β-sheet peptide tetramer byvirtue of the presence of relatively well-defined, downfield shifted αHand NH resonances. Assuming a similar β-sheet alignment and number (13to 16) of downfield shifted αH resonances as found in any nativeα-chemokine and normalizing to the aromatic resonance area (10 protons),it was estimated that this αH resonance area represents a fully foldedβpep-1. Compared to betabellin 14D, NMR resonances of the β-sheet foldedstate for betabellin 14D were broader than would be expected for a dimerof its size, indicating formation of larger aggregates. Moreover,betabellin 14D folding was also apparently not as compact as that foundin βpep-1.

NMR spectra for βpep-5, βpep-8 and βpep-11 also showed downfield shiftedαH and NH resonances, which indicates β-sheet formation. While CD datasuggested greater than 90% β-sheet structure for βpep-8 and βpep-11, NMRdata suggested somewhat less. CD data, however, would give evidence forsignificant β-sheet structure even if it were highly transient in amolten globule-like or non-compact state. The presence of thesedownfield shifted NMR αH resonances, therefore, indicated populations ofrelatively well-formed β-sheet conformation which are in slow chemicalexchange (600 MHZ NMR chemical shift time scale) with “non-compact” or“unfolded” conformational states whose αH protons resonate more upfield.For βpep-11, downfield shifted NHs were present in D₂O for an extendedperiod of time (4 hours), supporting the idea of some structuralstability. Downfield shifted αH and NH resonances, however, were verybroad with an overall envelope half-height of about 500 Hz. Althoughthis resonance broadening could be the result of this exchange process,the possibility of intermediate exchange among similarly folded β-sheetconformations or exchange among aggregate states were also investigated.

EXAMPLE 4 Pulsed-Field Gradient (PFG) NMR Self-Diffusion Measurements ofβ-Peptide Series Peptides

Pulsed-field gradient (PFG) NMR diffusion measurements (Gibbs, et al. J.Magn. Reson 93:395-402, 1991) were performed on βpep peptides to assessself-association properties. Pulsed field gradient (PFG) NMRself-diffusion measurements were made on a Bruker AMX-600 using a GRASPgradient unit. NMR spectra for measurement of diffusion coefficients, D,were acquired using a 5 mm triple-resonance probe equipped with anactively shielded z-gradient coil. The maximum magnitude of the gradientwas calibrated by using the standard manufacturer's (Bruker) procedureand was found to be 60 G/cm which is consistent with the value of 61G/cm obtained from analysis of PFG data on water using its knowndiffusion constant. The PFG longitudinal eddy-current delaypulse-sequence was used for all self-diffusion measurements which wereperformed in D₂O over the temperature range 275° K to 310° K. Peptideconcentrations ranged from 3 mg/mL to 10 mg/mL.

For unrestricted diffusion of a molecule in an isotropic liquid, the PFGNMR signal amplitude normalized to the signal obtained in the absence ofgradient pulses is related to D by:

R=exp[−γ² g ²δ² D(Δ−δ/3)]

where γ is the gyromagnetic ratio of the observed nucleus; g and δ arethe magnitude and duration of the magnetic field-gradient pulses,respectively, and Δ is the time between the gradient pulses. For thesestudies, experimental conditions were: δ=4 ms, g=1 to 45 G/cm, Δ=34.2ms, and the longitudinal eddy-current delay T_(c)=100 ms. Each diffusionconstant was determined from a series of 15 one dimensional PFG spectraacquired using different g values. Experimental decay curves wereapproximated as single exponentials.

Diffusion coefficients for peptides were calibrated by performing thesame PFG NMR self-diffusion measurements on globular polypeptideslysozyme, ribonuclease and ubiquitin. Here, PFG measurements yielded Dvalues at 20° C. of 10.1×10⁻⁷ cm²/s for lysozyme, 10.2×10⁻⁷ cm²/s forribonuclease, and 14.3×10⁻⁷ cm²/s for ubiquitin. These D values werewithin those values in the literature: 10.6×10⁻⁷ cm²/s for lysozymeobtained from light scattering by extrapolation to infinite dilution;10.7×10⁻⁷ cm²/s for ribonuclease also obtained from light scattering byextrapolation to infinite dilution, and 14.9×10⁻⁷ cm²/s for ubiquitin(Altieri et al., J. Am. Chem. Soc. 117: 7566-7567, 1995) obtained byusing similar PFG NMR measurements. This relatively good agreement indiffusion coefficients indicated that the PFG longitudinal eddy-currentdelay pulse sequence allows derivation of accurate diffusion constantvalues.

The Stokes-Einstein relationship D=k_(B)T/6πηR was used to relate D tothe macro-molecular radius, R, which was considered to be proportionalto the square root of the apparent molecular weight, M_(app) ^(1/2)(Cantor et al. 1980. The behavior of biological macromolecules.Biophysical Chemistry, part III. New York: W.H. Freeman. pp. 979-1039).Therefore, D is proportional to M_(app) ^(−1/2). From these simplerelationships, the ratio D_(dimer)/D_(monomer) was 0.71, which is veryclose to 0.72 theoretically predicted for a two sphere dimer (Wills, etal. J. Phys. Chem. 85: 3978-3984, 1981). Use of the Stokes-Einsteinrelationship is specifically derived for a hard sphere, and although theactual molecular shape of each peptide aggregate would affect thediffusion coefficient, the maximum change in the ratioD_(aggregate)/D_(monomer) would be about 20% in case of an improbablelinear peptide tetramer. M_(app) for peptides was calculated using Dvalues of lysozyme, ribonuclease and ubiquitin as standards for monomersof known molecular weight.

The temperature dependence of diffusion coefficients is plotted in FIG.4. By calibrating with diffusion data on lysozyme, ubiquitin, andribonuclease, the average molecular weight for these peptides at 30° C.was determined: βpep-5=12,750; βpep-8=9,490; βpep-11=41,200, andβpep-1=13,700. Moreover, for βpep-5, βpep-8 and βpep-1, the temperaturedependence was linear and followed the activation energy expected forthe self-diffusion of water. However, the aggregate state distributionchanged for βpep-8 below 10° C. and for βpep-11 over any temperaturerange investigated. For βpep-5 and βpep-1, the average molecular weightderived from these diffusion constants divided by the calculated monomermolecular weight (Table 1) yielded a ratio of 3.6. Since the temperaturedependence of the diffusion constants was linear, the aggregate statedistribution is unchanged, suggesting that a single aggregate state waspresent. βpep-1 showed compact β-sheet structure; βpep-5 was less clear.Given the fact that these apparent molecular weights were not beingcorrected for shape and electrostatic effects, the derived aggregatemolecular weights can be subject to some variation. Therefore, βpep-1may fold as a compact tetramer, and while βpep-5 also may tetrameric,there may be some distribution of monomer-dimer-tetramer. In general,these peptides formed tetramers.

A distribution of aggregate states was present in βpep-8 and βpep-11.The βpep-8 slope remained linear between 10° C. and 40° C. and deviatedfrom linearity at lower temperatures. The ratio of its apparentmolecular weight to its calculated monomer molecular weight (seeTable 1) was 2.4 up to 10° C. and changed to 3.7 by 2° C. These datasuggested that βpep-8 is on average a dimer and increased itsaggregation state, probably to a tetramer, at lower temperatures.βpep-11 showed most unusual diffusion characteristics. Its aggregatestate distribution continually changed with temperature. At lowertemperature, its average molecular weight was that of a tetramer, whileat higher temperatures, large aggregates upwards of octamers appeared toform.

EXAMPLE 5 Endotoxin Neutralizing Activity and Bactericidal Activity ofβpep-5, βpep-8, βpep-11 and βpep-1

The peptides were tested for their ability to neutralize endotoxin.Quantitative endotoxin activities for these peptides were measured byLimulus amoebocyte lysate assay.

Bacteria: P. aeruginosa type 1 is a clinical isolate maintained in thelaboratory. The isolate remains a smooth strain and was serotyped by thescheme of Homma. The Pseudomonas strain was maintained by monthlytransfer on blood agar plates.

Purification of B/PI: B/PI was purified in three column-chromatographysteps. In the final step, the sample was applied to a 1×180 cm molecularsieving column of Toyopearl HW55S (TosoHaas, Philadelphia Pa.) which hadbeen equilibrated with 0.05 M glycine buffer (pH 2.5) containing 0.5MNaCl. Polypeptide concentration was determined according to Hartree(Analytical Biochem. 48:422-427 (1972)). Purity was confirmed byvisualization of a SDS polyacrylamide gel following electrophoresis of 1μg of purified BP55 polypeptide and silver staining of the gel usingtechniques well known in the art.

Limulus amoebocyte lysate assay: The ability of synthetic peptides toneutralize endotoxin was detected with the E-TOXATE kit manufactured bySigma Chemicals (St. Louis, Mo.). The concentration of peptide requiredto completely inhibit the coagulation of Limulus amoebocyte lysatedriven by 0.04 unit (or 0.01 ng) of E. coli 055:B5 LPS was determined bydose response and as outlined in technical bulletin No. 210 of SigmaChemicals. Peptide or B/PI was incubated with LPS at room temperaturefor 5 min in 100 ul of a 1:2 dilution of pyrogen-free saline (pH 6.4).The reaction was started by addition of 100 ul of amoebocyte lysate andthe final volume was 200 ul. Assuming a 10,000 molecular weight for LPS,the approximate molar ration of B/PI:LPS at neutralization was 3000 to1.

The peptides were tested according to the method provided above. PF4(see FIG. 1) was used as a negative control and demonstrated noendotoxin neutralizing activity. BG38L corresponds to amino acids 86-108of B/PI and domains within B/PI have been shown to demonstrate endotoxinneutralizing activity. BG38L was used as a positive control in theseexperiments. The endotoxin neutralizing data is provided below:

TABLE 3 Endotoxin Neutralizing Activity Endotoxin Neutralizing^(a)Activity Peptide 5.0 × 10⁻⁵ M peptide βpep-5 70.3, n = 3 βpep-8100ED^(c) 1.2 × 10⁻⁶M, n = 7 βpep-11 100ED^(c) 1.2 × 10⁻⁶ (29.5), n = 5βpep-1 42.8, n = 3 PF4 0 ED^(c) 5.0 × 10⁻⁵ (23), n = 1 BG38 34.4, n = 5^(a)% neutralization of 0.02U(0.2 pg of endotoxin from E. coli 055:B5)^(b)% killing of 5 × 10⁵ P. aeruginosa ^(c)Effective M dose (%)

This data demonstrates that the B peptides of this invention wereeffective at neutralizing endotoxin activity.

EXAMPLE 6 Assay to Determine Bactericidal Activity

The peptides can be assayed for bactericidal activity against a varietyof organisms such as Pseudomonas aeruginosa type 1, P. cepacia ATCC25603, E. coli B, and Staphylococcus aureus 502A by standard methods.

Pseudomonas aeruginosa type 1 is a clinical isolate. The isolate remainsa smooth strain and was serotyped by the scheme of Homma (Jpn. J. Exp.Med. 46:329, 1976). A rough strain, E. coli B and S. aureus 502A wereobtained from Paul Quie, University of Minnesota (Minneapolis, Minn.).The characteristics of the S. aureus strain have been described byShinefield et al. (Amer. J. Dis. Chil. 105:646, 1963). Pseudomonascepacia ATCC 25608 can be purchased from the ATCC. S. aureus and E. coliwere maintained on nutrient agar plates and the Pseudomonas strains weremaintained on blood agar plates.

Pyrogen-free solutions were used throughout the assay and all methodsthat follow. Log phase bacteria were prepared from a culture in brainheart infusion broth (Hovde et al. Infect. Immun. 54:142-148, 1986) andbacteria were washed and resuspended in saline with adjustment to anoptical density at 650 nm to provide a yield of about 3×10⁸ CFU/ml.Bacteria were diluted 1:10 in 0.08M citrate phosphate buffer, pH 5.6 orpH 7.0, for use in the assay. S. aureus rapidly lost viability in the pH5.6 buffer and was studied only at pH 7.0. Bactericidal activity wasdetermined by dose response over peptide concentrations ranging fromabout 1.2×10⁻⁴ M to abut 4.1×10⁻⁹ M. Bactericidal activity wasdetermined by dose response, preferably determining LD₅₀ by linearregression.

Results are as follows:

TABLE 4 Endotoxin Neutralizing Activity and Bactericidal Activity ofβpeptides Endotoxin.Neutralization.^(a) Bactericidal Activity^(b)Peptide 5.0 × 10⁻⁶M Peptide 1.2 × 10⁻⁷ M Peptide βpep-1 42.8, n = 332.0, n = 3 βpep-2 55.3, n = 5 18.8, n = 6 βpep-3 79.4, n = 10 24.9, n =3 βpep-4 14.7, n = 3 37.6, n = 3 βpep-5 70.3, n = 3 7.4, n = 7 βpep-679.3, n = 3 8.2, n = 2 βpep-7 77.7, n = 2 39.1, n = 2 βpep-8 100 Ed^(c)1.2 × 10⁻⁶(77.9) 0 Ed^(c) 9.6 × 10⁻⁷ n = 7 (28.1), n = 3 βpep-9 100Ed^(c) 1.2 × 10⁻⁶(32.3) 0 Ed^(c) 9.6 × 10⁻⁷ n = 9 (47.4), n = 11 βpep-10100 Ed^(c) 1.2 × 10⁻⁶(25.2) 0 Ed^(c) 9.6 × 10⁻⁷ n = 8 (20.9), n = 7βpep-11 100 Ed^(c) 1.2 × 10⁻⁶(29.5) 0 Ed^(c) 9.6 × 10⁻⁷ n = 5 (48.0), n= 4 βpep-12 100 Ed^(c) 1.2 × 10⁻⁶(29.5) 0 Ed^(c) 9.6 × 10⁻⁷ n = 3(49.1), n = 5 βpep-13 100 Ed^(c) 1.2 × 10⁻⁶(22.5) 0 Ed^(c) 9.6 × 10⁻⁷ n= 1 (19.8), n = 3 βpep-14 38.8 n = 1 0 Ed^(c) 9.6 × 10⁻⁷(39.9), n = 3βpep-15 48.1 n = 1 0 Ed^(c) 9.6 × 10⁻⁷(23.9), n = 3 βpep-16 100 Ed^(c)1.2 × 10⁻⁶(16.5) 0 Ed^(c) 9.6 × 10⁻⁷(24.3), n = 3 n = 3 βpep-17 100Ed^(c) 1.2 × 10⁻⁶(28.9) 0 Ed^(c) 9.6 × 10⁻⁷(40.1), n = 4 n = 3 βpep-18100 Ed^(c) 1.2 × 10⁻⁶(30.2), n = 1 18.8, n = 1 βpep-19 0 Ed^(c) 1.6 ×10⁻⁵(12.4), n = 2 62.5, n = 2 βpep-20 100 Ed^(c) 1.2 × 10⁻⁶(17.1), n = 10 Ed^(c) 4.8 × 10⁻⁷(0.0), n = 4 βpep-21 100 Ed^(c) 1.2 × 10⁻⁶(40.3), n =1 13.0, n = 1 βpep-22 100 Ed^(c) 1.2 × 10⁻⁶(21.3), n = 4 35.5, n = 3βpep-23 100 Ed^(c) 1.2 × 10⁻⁶(65.7), n = 3 0 Ed^(c) 4.8 × 10⁻⁷(45.8), n= 3 PF4 0 Ed^(c) 5.0 × 10⁻⁵(0), n = 2 0 Ed^(c) 1.5 × 10⁻⁶(19), n = 2^(a)= % neutralization of 0.02U (0.2 pg of endotoxin from E. coli 055:B5^(b)= % killing of 5 × 105 P. aeruginosa ^(c)= Effective M dose (%)The best endotoxin neutralizing activity was identified for βpep-8 andβpep-23. The peptides tested that had the greatest bactericidal activityincluded βpep-19, βpep-7, βpep-4, βpep-22 and βpep-1.

EXAMPLE 7 βpep-3 is an Endotoxin Agonist

All peptides, BG22 (a 27mer, BPI aa 82-108), βpep-3 (SEQ ID NO:6) andBG16 (control peptide derived from a portion of BPI exclusive of the LPsbinding domain, aa 170-199), were synthesized at the University ofMinnesota microchemical facility using a mMillgren bioresearch 9600peptide synthesizer as described (Mayo et al. Protein Science5:1301-1315, 1996). Amino acid composition of the peptides was confirmedby gas-phase Edman degradation, using an Applied Biosystems 470Agas-phase sequenator with a reverse-phase C18 column. Purified peptideswere resuspended in pyrogen-free PBS prior to their use in allexperiments. The structure of βpep-3 was confirmed by two dimensionalnuclear magnetic resonance and circular dichroism spectroscopy asdescribed by Ilyine E. et al. (Biochem J. 306:407-419, 1995).

BG22 82-108BPI NANIKISGKWKAQKRFLKMSGNFDLSI (SEQ ID NO:31) Bpep-3BPI/IL-8 HIKELQV KWKAQKRFLKMSI IVKLNDGRELSL D (SEQ ID NO:3) BG16170-199BPI MNSQVCEKVTNSVSSKLQPYFQTLPVMTKI (SEQ ID NO:32)

Underlined amino acid sequence represents the LPS binding domain of BPI

italicized sequences are derived from the amino acid residuesresponsible for initiation of β-turning in native IL-8.

LPS derived from P. aeruginosa and E. coli 0111:B4 or 055:B5 waspurchased from List Biological Laboratories (Campbell, Calif.) andstored at 4° C. until use. A commercially available chromogenic Limulusamoebocyte lysate test (Whittaker, Walkersville, Md.) was used tomeasure endotoxin levels in all solutions and serum samples. Endotoxinlevels in peptide solutions were determined to be less than about 0.1ng/ml. The capacity of BG22, βpep-3 and BG16 to neutralize E. coli055:B5 endotoxin in vitro was determined by mixing 0.01 ng of LPS with5×10⁻⁶M of each peptide and results were compared to that observed for0.01 ng of LPS alone.

5×10⁻⁵ RAW 264.7 cells were placed in each well of a 24-well cultureplate and allowed to adhere. Immediately before LPS stimulation, mediumwas removed and cells were washed in serum-free Dulbecco's modifiedEagle's medium (DMDM). Cells were incubated at 37° C. in a 10% CO₂atmosphere for 3 hours with: 1) DMEM alone, 2) E. coli 0111:B4 LPSalone, or 3) E. coli 0111:B4 LPS preincubated with BG22, βpep-3 or BG16.The concentration of LPS used in all groups was 200 ng/ml. To avoidnon-specific inhibition of LPS-induced TNF-α secretion, 10⁻⁵ M of eachpeptide was used in the experimental wells. At the end of the incubationperiod, macrophage supernatants were collected and assayed for TNF-αconcentration.

TNF-α concentrations were measured as described by Battafarano et al.(supra). Briefly, the TNF-α sensitive cell line WEHI was used in allassays of either RAW cell supernatant (in vitro) or serum (in vivoanimal model). Concentrations were extrapolated from a standard curvebased on dilutions of purified TNF-α (Genzyme, Cambridge, Mass.), andall samples were examined in triplicate. Viability of the WEHI cells wasassessed by measuring the extent of cellular crystal violet uptake byspectrophotometry at 590 nm. In this assay, absorbance was inverselycorrelated with cell death and lysis resulting from exposure of WEHIcells to TNF-α. The amount of TNF-α in each experimental sample wasextrapolated from the standard curve.

To study endotoxemia in a mammal, 500 μg of each peptide was added to 8μg of Pseudomonas aeruginosa LPS in 2.0 ml of pyrogen-free phosphatebuffered saline (PBS) ex vivo, and incubated for 30 minutes oil a shakerat room temperature. Each mouse was injected via tail vein with 0.5 mlof the mixture (125 μg of BG22, BG16, or βpep-3 and 2 μg of LPS permouse). Mice were euthanized at 30, 60, 90 and 120 minutes afterinjection and serum collected just prior to death was analyzed forendotoxin levels and TNF-α concentration.

All TNF-α and endotoxin measurements in duplicate or triplicate in eachexperiment. In vivo experiments were performed twice to confirmreproducibility. The results obtained were compared using the unpairedStudent's/t-test.

FIG. 5 provides the results of studies to determine the capacity ofβpep-3 to diminish LPS-induced secretion of TNF-α in vitro by RAW 264.7cells. Both BG22 and βpep-3 neutralized endotoxin, compared to BG16which lacked any endotoxin neutralizing capabilities (37% and 81% vs.0%, respectively; p<0.05). Interestingly, βpep-3 neutralized LPS to asignificantly greater degree than BG22 (see Table 5: p<0.05). Of note,preincubation with βpep-3 caused a significantly greater decrease inTNF-α secretion, compared to that which was observed for BG22 (p=0.04,FIG. 5).

Preincubation of LPS with peptides BG22 and βpep-3 prior to intravenous(IV) challenge led to a similar, significant dimunution in serumendotoxin levels at all time points measured (30, 60, 90, and 120minutes), compared to BG16 or LPS without peptide (p<0.05, Table 6).Similarly, preincubation of either BG22 or βpep-3 with LPS before ivinjection resulted in decreased serum TNF-α levels at 90 minutespost-challenge, compared to mice receiving LPS without peptide (p<0.05,FIG. 6). Either BG22 or βpep-3 injection resulted in decreased serumTNF-α levels compared to mice receiving LPS without peptide. Althoughthere appeared to be greater inhibition of TNF-α in mice receiving theβpep-3 versus BG22, the difference was not statistically significant.

The endotoxin binding domain of PBI forms an amphipathic β-turn withalternating charged and hydrophobic amino acid residues. This secondarystructure can be important to effect binding interactions between thepeptides and LPS. βpep-3 folds with a β-turn at physiologic pH.

Both βpep-3 and BG22 bound to endotoxin but βpep-3 was significantlymore effective in neutralizing endotoxin in vitro, compared with BG22.In fact, this finding correlated with the ability of βpep to moreeffectively inhibit LPS induced TNF-α secretion by macrophages in vitro.Without intending to limit the scope of this invention, it seems likelythat the factor that may account for the enhanced anti-endotoxinproperties of βpep-3 in vitro is its folding such that it more closelyresembles the LPS binding domains of the native anti-LPS proteins.

Both BG22 and βpep-3 demonstrated similar efficacy in vivo, as evidencedby a reduction in circulating endotoxin and diminished TNF-α secretionduring murine endotoxemia. Several factors may account for the relativeinability to notice a significant difference between these peptides inthe murine model such as the competition for LPS binding by endogenousproteins (including, for example, LBP, lipopolysaccharide bindingprotein, and 2) rapid clearance or short half-life or small peptides.The binding affinity of βpep-3 can be enhanced via site-directed changesin amino acids and the biologic half-life of this peptide along withothers prepared by the methods of this invention can be increased viaconjugation of these peptides to larger, stable carrier proteinsincluding, for example, keyhole limpet hemocyanin.

TABLE 5 Determination of the capacity of anti-endotoxin peptides BG22and βpep-3 to neutralzine endotoxin in vitro. βpep-3 demonstratedenhaneed capacity to neutralize endotoxin comapred to BG22 or BG16.Peptide % Neutralization of Endotoxin BG22 37% βpep-3 81% (p < 0.05)BG16  0%

TABLE 6 Determination of the capacity of anti-endotoxin peptides BG22and βpep-3 to diminish endotoxemia in vivo. Both BG22 and βpep-3significantly diminish endotoxemia compared to control peptide BG16.Minutes after LPS/Peptide Injection 30 60 90 120 BG22     7*    28*   73*    47* βpep-3    11*    37*    63*    66* BG16 >10,000  2,394 5,599 >10,000 LPS >10,000 >10,000 >10,000 >10,000 *p < 0.05

EXAMPLE 8 βPeptide Series Peptides as Angiogenesis Inhibitors

To test the hypothesis that angiostatic factors can prevent or inhibitangiogenesis medicated downregulation of endothelial adhesion molecules,angiogenesis and endothelial cell proliferation, PF4, one of the mostpotent angiogenesis inhibitors, and its related βpep-peptides weretested for their effects on endothelial adhesion molecule expressionfrom endothelial cells (EC). Like the native PF4, βpep-14 and βpep-16peptides were found to be angiostatic as determined by measurement of ECproliferation in vitro (Table 7). We also studied the effect of thepeptides on expression of intercellular adhesion molecule-1 (ICAM-1)since in earlier studies it was demonstrated, by the use of blockingantibodies, that ICAM-1/LFA-1 interaction was most important inleukocyte/EC adhesion and extravasation.

In a first series of experiments, PF4 and βpep peptides were tested fortheir ability to prevent bFGF (fibroblast growth factor) mediateddownregulation of ICAM-1. It was found that inhibition of angiogenesisand endothelial cell proliferation resulted in a complete blockade ofbFGF mediated ICAM-1 downregulation. A 3-day preincubation of EC with 10μg/ml bFGF resulted in a marked modulation of ICAM-1. Simultaneously 100μg/ml of each PF4, βpep-14, βpep-16, or medium was added. Mean ICAM-1fluorescence intensity values were determined. The addition of 100 μg/mlPF4 enhanced the expression of ICAM-1. Simultaneous addition of bFGF andPF4 did not result in the loss of ICAM-1 expression. Also, the additionof the PF4 related peptides βpep-14 and βpep-16 resulted in a completeblock of bFGF mediated downregulation.

Since the in vivo situation of tumor associated EC involves the lowexpression or even absence of ICAM-1, the next set of experiments aimedto study the ability to reinduce ICAM-1 expression after bFBFpreincubation. It had been demonstrated previously that the longevity ofthe bFGF mediated ICAM-1 downregulation is at least 7 days. Treatment ofEC expressing downregulated ICAM-1 levels with 100 μg/ml PF4 resulted,even in the presence of bFGF, in reinduction of ICAM-1. βpep-14 andβpep-16 showed similar results. In these experiments, HUVEC werepretreated for 3 days with bFGF, subsequently PF4 was added for 3 daysand, where indicated in the last 16 hours of culture 4 ng/ml TNFα wasadded. Human umbilical vein derived endothelial cells (HUVEC) wereharvested from normal human umbilical cords by perfusion with 0.125%trypsin/EDTA as described previously. Cells were cultured in fibronectin(FN) coated tissue culture flasks in culture medium (RPMI-1640 with 20%human serum (HS), supplemented with 21 mM glutamine and 100 U/mlpenicillin and 0.1 mg/ml streptomycin). Immunofluorescence usingindirect PE-conjugated reagents required three separate incubations.1×10⁵ EC were fixed for 1 hour in 1% paraformaldehyde, resuspended in 20μl appropriately diluted Mab and incubated for 1 hour on ice.Subsequently, cells were washed two times in PBS/BSA (0.1%) andincubated for another 30 minutes with biotinylated rabbit-anti-mouse Ig(Dako, Glostrup, Denmark). After another 2 washings, cells wereincubated with streptavidin-phycoerythrin conjugate (Dako). Stainedcells were analyzed on a FACScan flowcytometer. Data analysis wasperformed using PCLysys software (Becton Dickinson, Mountain View,Calif.). Statistical significance of observed differences was determinedusing the Student's t-test.

The anergy of EC to stimulation with inflammatory cytokines was thesubject of a next series of experiments. For these experiments, HUVEC(human vascular endothelial cells) were pretreated with 10 ng/ml bFGFfor 3 days. Subsequently, cells are subcultured for 3 days with 100ng/ml bFGF in the presence of PF4. For the last 16 hours of the culture4 ng/ml TNFα was added to induce upregulation of ICAM-1. The decreasedinflammatory response of angiogenic stimulated EC was found to beovercome by simultaneous treatment with PF4 and similar results werefound for βpep-14 and βpep-16. The regulation of ICAM-1 at the proteinlevel was confirmed in Northern blot analysis for detection of ICAM-1message. In these experiments, HUVEC were cultured for 3 days with bFGFand treated for the last 4 and 24 hours with PF4 (100 μg/ml). TNFα wasadded 2 hours before isolation of RNA. RNA from a subconfluent ECcultures (75 cm² Petri-dishes) incubated with bFGF for differenttime-points was isolated using an RNA-zol kit (Campro Scientific,Houston, Tex.). Total RNA (10 μg) for each sample was separated in a0.8% formaldehyde-denaturing gel, transferred to nitrocellulose (HybondN+, Amersham International, Amersham, UK) and hybridized to a³²P-labelled 1.9 Kb c-DNA probe, containing the functional sequence ofthe human ICAM-1 gene (a gift from Dr. B. Seed). Membranes were washedat a high stringency in 0.2×SSC, 0.1% SDS at 50° C. for 1 hour Filterswere exposed to X-ray films (Kodak X-omat, Eastman Kodak Company,Rochester, N.Y.) using an intensifying screen at −80° C. for not lessthan 12 hr. Autoradiograms were subjected to scanning using a laserdensitometer (Model GS670, Bio-Rad, Hercules, Calif.) and data wereanalysed with the Molecular Analyst PC™ software. The intensity of themajor ICAM-1 mRNA transcript was normalized with respect to actin mRNAexpression used as a control.

Functional impact for the observed phenomena was provided byleukocyte/EC adhesion assays as described earlier (Griffioen et al.Cancer Res. 56:1111-1117, 1996). The bFGF mediated inhibition ofleukocyte adhesion to cultured HUVEC was completely abolished in thepresence of PF4 or related peptides. TNF mediated upregulation ofadhesion to bFGF preincubated HUVEC in the presence of PF4 was similarto the adhesion to TNF treated control cells. PHA-activated peripheralblood T lymphocyte were adhered for 1 hour at 37° C. to TNF-α (4 ng/ml),bFGF (10 ng/ml)+PF4 (100 μg/ml) treated, or control (HUVEC).Non-adhering cells were removed and adhered cells were enumerated by aninverted microscope. Values of one representative experiment out ofthree are expressed as numbers of adhered cells per high power field.Statistical significance is determined by the Student's t-test.

These results indicate that the inhibition of angiogenesis andendothelial cell proliferation, which has been demonstrated to preventoutgrowth of solid tumors and metasteses, is able to overcome the downregulation of adhesion molecules and the anergy upon stimulation withinflammatory cytokines. In experiments to document the effect of otherinhibitors of angiogenesis the same results were found forthrombospondin-1 and IP-10.

However, the metalloproteinase inhibitor BB-94 (batimastat) andthalidomide, which do not affect EC growth in vitro, did not affectICAM-1 expression. We concluded that the ICAM-1 regulation coincideswith the regulatory mechanisms involving EC growth. The present resultsindicate that adhesion molecules which are necessary for the formationof an efficient leukocyte infiltrate are not only under regulation ofangiogenic factors but are induced under conditions of angiogenesisinhibition. Specific inhibition of EC growth in vivo and regulation ofEAM is therefore a powerful tool in cancer therapy. Definition ofsynthetic non-endogenous active peptides (Such as βpep type peptides,including βpep-14) will contribute to this approach.

TABLE 7 INHIBITION OF EC-PROLIFERATION (³H)-THYMIDINE INCORPORATION BYDIFERENT ANTIOGENESIS INHIBITORS no bFGF 10 ng/ml bFGF expt 1 medium4044 ± 206 28815 ± 1007 PF4 (1 μg/ml) 4656 ± 456 28782 ± 815  PF4 (10μg/ml) 4066 ± 351 23868 ± 402  PF4 (100 μg/ml) 1651 ± 172 4655 ± 421expt 2 medium 14296 ± 2490 29079 ± 2506 βpep-14 (1 μg/ml) 14184 ± 177528695 ± 1062 βpep-14 (10 μg/ml)  9886 ± 2114 29530 ± 1608 βpep-14 (100μg/ml) 3774 ± 299 6585 ± 132 βpep-16 (1 μg/ml) 15039 ± 2020 35447 ± 2621βpep-16 (10 μg/ml) 11881 ± 2545 33663 ± 2572 βpep-16 (100 μg/ml) 4929 ±749 7852 ± 875 expt 2 medium 6780 ± 713 52808 ± 4092 PF4 (1 μg/ml)  6171± 1227 43524 ± 5318 PF4 (10 μg/ml) 3547 ± 317 8337 ± 704 PF4 (100 μg/ml) 947 ± 170 1654 ± 375 βpep-14 (1 μg/ml)  7214 ± 1668 48443 ± 2700βpep-14 (10 μg/ml) 6074 ± 899 52126 ± 1258 βpep-14 (100 μg/ml) 1062 ±325 7663 ± 715 βpep-16 (1 μg/ml) 7450 ± 737 47727 ± 447  βpep-16 (10μg/ml)  6148 ± 1370 44919 ± 2081 βpep-16 (100 μg/ml) 2669 ± 370 27071 ±3277 expt 3 medium ND 3432 ± 232 IP-10 (100 μg/ml) ND 725 ± 95 expt 4medium 18904 ± 1501 31954 ± 1220 TSP-1 (10 μg/ml) 8865 ± 639 22338 ±860  TSP-1 (25 μg/ml) 5565 ± 349 10267 ± 797 EC proliferation was measured using a ³[H]thymidine incorporation assay.EC were seeded in flatbottomed 96-well tissue culture plates (5000cells/well) and grown for 3 days, in culture medium. In some culturesthe proliferation of EC was enhanced by incubation with 10 ng/ml bFGF.During the last 6 hours of the assay, the culture was pulsed with 0.5μCi [methyl-³H]thymidine/well. Results are expressed as the arithmeticmean counts per minute (cpm) of triplicate cultures.

βpeptides 1-24 were tested in an endothelial cell proliferation assayusing ³H-thymidine incorporation. At least half of the peptides weresomewhat active at 2.6 micromolar at decreasing endothelial cell growth.These results are provided in FIG. 7. βpep-23 and βpep-24 were about 30%effective at 0.26 micromolar.

The peptides were also able to regulate inter-cellular adhesion molecule(ICAM) expression. This receptor is downregulated in tumors and agentsthat are effective at upregulating ICAM are potentially usefultherapeutic agents to control tumor growth. Those that were the mostanti-angiogenic appeared to be least effective at ICAM regulation. Thatthe βpeptides have the same or similar positive charge to mass ratiosbut do not share the same activities indicates that the peptides workspecifically. For example, βpep-8 demonstrates little cell proliferationactivity while βpep-24 was very good at controlling cell proliferation.Those skilled in the art will readily be able to use the assays providedhere and the βpep sequences disclosed herein, along with methods forproducing additional peptides according to this invention to identifypeptides with ICAM upregulating activity and peptides with endothelialcell proliferation activity without undue experimentation.

All references cited herein are incorporated by reference, in theirentirety, into this text. Although the invention has been described inthe context of particular embodiments, it is intended that the scope ofcoverage of the patent be limited only by reference to the followingclaims.

1-20. (canceled)
 21. A water soluble peptide selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 SEQ ID NO:20, SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23 SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 SEQ IDNO:28, SEQ ID NO:29, and SEQ ID NO:30. 22-32. (canceled)
 33. The peptideof claim 21 further comprising a pharmaceutically acceptable carrier.34. The peptide of claim 42 wherein said pharmaceutically acceptablecarrier is selected from the group consisting of isotonic saline,dimethylsulfoxide, alcohol, phosphate buffered saline, and otherbalanced salt solutions.
 35. The peptide of claim 21 conjugated to aprotein carrier.
 36. The peptide of claim 35 wherein said proteincarrier is selected from the group consisting of keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin.
 37. Amethod for treating a bacteria infection or endotoxic shock comprisingadministering to a mammal an amount of a pharmaceutical compositioneffective to inhibit the bacterial infection or neutralize endotoxin,wherein the pharmaceutical composition comprises the water solublepeptide of claim 21 and a pharmaceutically acceptable carrier.
 38. Amethod for inhibiting TNF-α levels in a mammal comprising administeringa therapeutically effective amount of a pharmaceutical compositioncomprising the water soluble peptide of claim 21 and a pharmaceuticallyacceptable carrier.
 39. A method for inhibiting endothelial cellproliferation comprising administering to a mammal an effective amountof a composition comprising the water soluble peptide of claim
 21. 40. Amethod for promoting inter-cellular adhesion molecule expressioncomprising administering to a mammal an effective amount of acomposition comprising the water soluble peptide of claim
 21. 41. Amethod for inhibiting angiogenesis in a cell culture, the methodcomprising contacting cells with an effective amount of a compositioncomprising the water soluble peptide of claim
 21. 42. A method forinhibiting angiogenesis in a mammal, the method comprising administeringto the mammal an effective amount of a composition comprising the watersoluble peptide of claim 21 and a pharmaceutically acceptable carrier.43. A method for inhibiting tumorigenesis in a mammal, the methodcomprising administering to the mammal an effective amount of acomposition comprising the water soluble peptide of claim 21 and apharmaceutically acceptable carrier.
 44. A method for inhibitingbacterial infection or endotoxic shock in a cell culture, the methodcomprising contacting cells with an effective amount of a compositioncomprising a the water soluble peptide of claim
 21. 45. A method forinhibiting TNF-α levels in a cell culture, the method comprisingcontacting cells with an effective amount of a composition comprising athe water soluble peptide of claim
 21. 46. A method for inhibitingendothelial cell proliferation in a cell culture, the method comprisingcontracting cells with an effective amount of a composition comprisingthe water soluble peptide of claim
 21. 47. A method for promotinginter-cellular adhesion molecule expression in a cell culture, themethod comprising contacting cells with an effective amount of acomposition comprising the water soluble peptide of claim
 21. 48. Amethod for inhibiting inter-cellular adhesion molecule expression downregulation in a mammal, the method comprising administering to themammal an effective amount of a composition comprising the water solublepeptide of claim
 21. 49. A method for inhibiting inter-cellular adhesionmolecule expression down regulation in a cell culture, the methodcomprising contacting cells with an effective amount of a compositioncomprising the water soluble peptide of claim
 21. 50. A method forinhibiting leukocyte/endothelial cell adhesion in a cell culture, themethod comprising contacting cells with an effective amount of acomposition comprising a the water soluble peptide of claim
 21. 51. Amethod of increasing leukocyte cell infiltration in a mammal, the methodcomprising administering to the mammal an effective amount of acomposition comprising a the water soluble peptide of claim 21.